Circadian clocks regulate a wide variety of cellular, physiological, and behavioral activities in almost all eukaryotic organisms. The importance of the circadian clock in human physiology and mental health is evident from its ubiquitous influence on a wide range of cellular and physiological processes. The malfunction of the clock is known to be associated with several forms of human sleep disorders and psychiatric illness. In Neurospora, one of the best understood eukaryotic circadian systems, FREQUENCY (FRQ) protein is an essential component of the circadian negative feedback loop. FRQ, like the PERIOD proteins in animal systems, is progressively phosphorylated and is degraded after extensive phosphorylation. We have shown that FRQ phosphorylation and dephosphorylation are mediated by several kinases (CK-1a, CKII, and CAMK-1) and phosphatases (PP1 and PP2A). The phosphorylation of FRQ promotes FRQ degradation, regulates FRQ/WC interaction, and controls the transcriptional represser activity of FRQ. FRQ is degraded through the ubiquitin-proteasome pathway and its ubiquitination is mediated by FWD-1, a F-box /WD40 repeat containing protein, which is a part of a SCF type E3 ubiquitin ligase. The conservation of kinases, phosphatases, and the degradation mechanism in regulating FRQ and Period proteins suggests a common foundation for diverse eukaryotic circadian clocks. In Specific Aim 1, we will determine the role of CK-1a in the Neurospora circadian clock. We will examine its role in FRQ phosphorylation and in the circadian negative feedback loop. In Specific Aim 2, we will determine how phosphorylation differentially regulates FRQ functions. We will map the in vivo FRQ phosphorylation sites by mass spectrometry analysis and determine their roles in regulating FRQ functions. We will also examine the regulation of FRQ phosphorylation events by the known FRQ kinases. In Specific Aim 3, we will determine how FRQ is degraded through the SCF(FWD-I) mediated ubiquitin-proteasome pathway. We will test the hypothesis that progressive FRQ phosphorylation is an important mechanism for period determination of the clock. We will also examine the role of COP9 in the circadian clock. Because of the conservation of the posttranslational regulations of eukaryotic clock proteins, our studies should provide important information relevant to the circadian systems in higher eukaryotic organisms.